cll-reactive t cell responses after stem cell
TRANSCRIPT
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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Development of Tumor-Reactive T Cells After Nonmyeloablative Allogeneic Hematopoietic
Stem Cell Transplant for Chronic Lymphocytic Leukemia
Tetsuya Nishida*,1 Michael Hudecek*,1 Ana Kostic,1 Marie Bleakley,1,2 Edus H. Warren,1,3
David Maloney,1,3 Rainer Storb,1,3 Stanley R. Riddell1,3
1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA,
Departments of Pediatrics2 and Medicine3, University of Washington, Seattle, WA, USA
* these authors contributed equally to this work
Acknowledgements: This work was supported by National Institutes of Health grants CA18029
and 114536 (S.R.R.), CA78902 (R.S.), CA15704 (Core), and the Lymphoma and Leukemia
Society. MH is a scholar of the German Research Foundation.
Correspondence to
Stanley R. Riddell, M.D.
Fred Hutchinson Cancer Research Center
1100 Fairview Avenue N., D3-100, Seattle, WA 98109
E-mail: [email protected]
Phone: +1-206-667-5249, Fax: +1-206-667-7983
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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Running Title: CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
Key words: chronic lymphocytic leukemia, graft-versus-tumor effect, graft-versus-host disease,
minor histocompatibility antigens, tumor-associated antigens
Conflict-of-interest disclosure: The authors have no relevant conflicts of interest to declare.
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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Statement of Translational Relevance
Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can cure
chemotherapy refractory chronic lymphocytic leukemia (CLL). A graft-versus-leukemia (GVL)
effect is required for tumor eradication after NM-HSCT, but the mechanisms responsible are not
well defined, and separating GVL from graft-versus-host disease (GVHD) is difficult. In a cohort
of NM-HSCT recipients, we investigated whether CLL-reactive T cells could be detected
post-transplant and correlated their presence with antitumor activity. T cells specific for CLL
developed with variable kinetics in all patients who achieved a complete remission, but not in
patients who failed to respond to NM-HSCT. CLL-reactive T cells were specific for recipient
minor histocompatibility antigens expressed by CLL and other recipient cells, but also antigens
that were only expressed by the malignant B cells. The molecular identification of minor
histocompatibility and tumor-associated antigens expressed on CLL may define targets for
adoptive T cell transfer or vaccination to augment GVL activity without GVHD.
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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Abstract
Purpose: Allogeneic NM-HSCT can result in durable remission of chronic lymphocytic
leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor
cells by T cells in the donor stem cell graft. We evaluated the development of cytotoxic T
lymphocytes (CTL) specific for CLL after NM-HSCT to determine if their presence correlated
with antitumor efficacy.
Experimental Design: Peripheral blood mononuclear cells obtained from twelve transplant
recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T cell
lines and CD8+ T cell clones were derived from these cultures and evaluated for lysis of donor
and recipient target cells including CLL. The presence and specificity of responses was
correlated with clinical outcomes.
Results: Eight of the 12 patients achieved remission or a major antitumor response and all eight
developed CD8+ and CD4+ T cells specific for antigens expressed by CLL. A clonal analysis of
the CD8+ T cell response identified T cells specific for multiple minor histocompatibility (H)
antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8+ T
cell response in some patients was also directed against non-shared tumor-specific antigens. By
contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after
NM-HSCT, despite the development of GVHD.
Conclusions: The development of a diverse T cell response specific for minor H and
tumor-associated antigens expressed by CLL predicts an effective GVL response after
NM-HSCT.
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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Introduction
Allogeneic hematopoietic stem cell transplantation (HSCT) can cure many hematological
malignancies, although graft-versus-host disease (GVHD) and relapse remain significant
obstacles. The efficacy of HSCT results from cytotoxic conditioning and a graft-versus-leukemia
(GVL) effect (1, 2). Myeloablative conditioning regimens that employ total body irradiation
and/or intensive chemotherapy exhibit potent antitumor activity, but are limited to young patients
due to nonhematopoietic toxicities. Allogeneic HSCT can be extended to older patients and those
with comorbidities using reduced intensity nonmyeloablative conditioning regimens that provide
less antitumor activity but immunosuppress the recipient sufficiently to allow engraftment of
donor hematopoietic cells and enable a GVL effect (3-7). Nonmyeloablative HSCT (NM-HSCT)
leads to remission in a subset of patients with refractory indolent hematologic malignancies
including chronic lymphocytic leukemia (CLL) (8-17). The eradication of CLL after NM-HSCT
is associated with GVHD, and presumed to be a consequence of T cell recognition of
alloantigens expressed by leukemic cells (18). However, many patients do not respond to
NM-HSCT despite developing GVHD and others respond without significant GVHD. Thus, the
basis for a successful GVL effect remains poorly defined in individual patients.
CLL is amenable to studies of the GVL effect because leukemia cells can be obtained
from most patients, and induced to become efficient antigen presenting cells (APC) by
stimulation through CD40 (19-21). Here, we used recipient CD40L stimulated CLL as APC to
isolate donor T cells that were specific for CLL after NM-HSCT. CD8+ and CD4+ T cells that
recognized multiple minor H antigens expressed on recipient CLL were isolated from all patients
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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who achieved or maintained a complete remission (CR) after NM-HSCT. In addition, CD8+ T
cell clones that recognized recipient CLL but not EBV-transformed B cells were isolated from
responding patients, suggesting a component of the response is directed against tumor-specific
determinants. Despite the development of GVHD and high levels of donor T cell chimerism,
CLL-specific T cells were not detected in recipients with persistent or progressive leukemia.
These results demonstrate that the specificities of the T cell responses that develop after
allogeneic NM-HSCT are critical in determining antitumor efficacy, and illustrate the potential to
manipulate T cell reactivity to target antigens expressed selectively by tumor cells to improve
outcome.
Materials and Methods
Patient and Donor Eligibility
Patients with CLL who failed to meet National Cancer Institute (NCI) Working Group
Criteria for complete or partial response (22) after therapy with a regimen containing fludarabine,
or who relapsed within 12 months after completing fludarabine, and had an HLA-A, -B, -C,
-DRB1, and –DQB1 matched related or unrelated donor were eligible. Exclusion criteria
included central nervous system involvement, history of malignancy other than CLL within the
past 5 years, Karnofsky score less than 60%, and severe cardiovascular, pulmonary, or hepatic
dysfunction. Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood was the
source of stem cells. All patients and their related donors signed consent forms approved by the
FHCRC institutional review board. Informed consent was obtained from the unrelated donors
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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according to National Marrow Donor Program (NMDP) regulations.
Treatment Plan
Patients were conditioned for transplantation with fludarabine 30 mg/m2/day on days –4
to –2, and 200 cGy of total body irradiation on day 0. The patients received immunosuppression
with cyclosporine or tacrolimus given orally every 12 hours beginning on day -3; and
mycophenolate mofetil given beginning on day 0 every 12 hours to recipients with related donors,
and every 8 hours to recipients with unrelated donors (16). Analysis of donor/recipient
chimerism in peripheral blood CD3+ and CD33+ cells was performed post-transplant on
approximately day 28, 56, and 84; at 6, 12, 18 months; and then annually.
Disease Response and GVHD Assessment
Disease response was assessed using NCI Working Group Criteria (22). Patients
underwent evaluation for minimal residual CLL by flow cytometry of peripheral blood and bone
marrow. Diagnosis and grading of acute and chronic GVHD were performed according to
established criteria (23, 24).
Cell Lines
Epstein-Barr virus transformed B-cells (B-LCL) were generated from patients and donors
as described (25). B-LCL and CLL from individuals of known HLA types were used as targets to
determine the HLA-restricting allele and the frequency of individual minor H or
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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tumor-associated antigens. Fibroblast cell lines were generated and maintained as described (25).
Isolation of CLL-Reactive T Cell Lines and Clones
PBMC were isolated by centrifugation over Ficoll-Hypaque and cryopreserved. NIH 3T3
cells transfected with human CD40L (tCD40L) were a gift of Dr. J. Schultze and used to
generate CD40 activated CLL cells (CD40-CLL) as described (26). After 3 days of activation, an
aliquot of cells was analyzed for expression of CD5, CD19, HLA-class I, HLA-class II, CD80,
CD86, CD54 and CD58 to determine purity and confirm activation through CD40. A second
aliquot of CD40-CLL was γ-irradiated and used as APC to stimulate post-transplant PBMC.
When further expansion of CLL cells was required, CD40-CLL were transferred to fresh tCD40L
every 3-4 days.
To generate T cell lines, PBMC (3x106/well) obtained from the recipient at intervals
post-transplant were stimulated with γ-irradiated (35 Gy) recipient CD40-CLL (0.75 x106/well)
in RPMI, 10% human serum, 2 mM L-glutamine and 1% penicillin-streptomycin. Cultures were
restimulated after seven days with γ-irradiated recipient CD40-CLL at a responder to stimulator
ratio of 4:1. The medium was supplemented with IL-7 (10 ng/mL on day 0 of each stimulation)
and IL-2 (2 U/mL) on day 3 after the first stimulation, and then 10 U/mL on days 1 and 4 of the
second stimulation. After 2 weeks, T cell lines were tested for cytotoxic activity against
recipient- and donor-derived B-LCL and recipient CD40-CLL.
CD8+ and CD4+ T cells that secreted IFN-γ after stimulation with CD40-CLL were
sort-purified using IFN-γ capture. An aliquot (1x106 cells) of each of the T cell lines was
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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co-cultured with CD40-CLL (1-2 x 106) in media containing 20 U/ml IL-2 at 37℃ for 4 hours
and then processed as described (27, 28). CD4+ and CD8+ IFN-γ+ cells were sorted and then
expanded by stimulation with anti-CD3 monoclonal antibody or cloned at 0.5 cells/well in
96-well round bottom plates with anti-CD3 monoclonal antibody (30 ng/ml) as described (25,
29). In selected cases γ-irradiated recipient CD40-CLL (2x103 cells/well) were used for
stimulation in cloning cultures in place of anti-CD3 monoclonal antibody. After 12-14 days,
cloning wells with growth were tested for cytotoxicity against recipient and donor B-LCL to
identify T cell clones specific for recipient minor H antigens. In selected cases, recipient CLL
cells were included as target cells during screening to identify T cell clones specific for putative
tumor-associated antigens expressed by CLL but not nonmalignant B cells.
Cytotoxicity Assays and ELISA
Target cells, which included B-LCL, CD40-CLL, primary CLL, and fibroblasts, were
labeled with 51Cr for 2 hours, washed twice, plated in triplicate at 1-2 x 103 cells/well with
effector cells at various effector to target (E/T) ratios. When less than 75% of the recipient
PBMC activated with CD40 expressed CD5 and CD19, the CD5+ CD19+ cells were sorted using
a FACSVantage cell sorter (Becton Dickinson) prior to cytotoxicity assays. Fibroblasts were
treated with 100 U/ml IFN-γ for 48 hours before use as targets. In some experiments,
Cr51-labeled target cells were pulsed with peptides (10 μg/ml) for 30 minutes, and then washed
prior to use in the assay. Supernatants were harvested for γ-counting after a 4-hour incubation of
effector and target cells, and specific lysis calculated using the standard formula.
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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For IFN-γ ELISA, target and effector cells were washed twice and plated in triplicate at
an E/T ratio of 3:1. After a 24 hour incubation, the supernatant was collected and analyzed for
IFN-γ using a primary and a biotinylated secondary anti human IFN-γ antibody (Pierce
Biotechnologies, Rockford, IL) according to the manufacturer’s instructions. Color development
was performed with streptavidin-conjugated HRP and TMP (Sigma-Aldrich, St. Louis, MO), and
optical density determined using a Thermo Multiskan EX reader (Thermo Scientific, Waltham,
MA). A standard curve was obtained with serial dilutions of recombinant human IFN-γ with a
potency of 2.0 x 104 IU/μg (R&D Systems, Minneapolis, MN).
Flow cytometry
T cells, primary CLL and CD40-CLL were stained with one or more of the following
monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE):
CD3, CD4, CD5, CD8, CD19, CD40, CD54, CD58, CD80, CD86, HLA-class I, HLA-DR and
isotype matched control antibodies (BD Pharmingen). Flow cytometric analysis was performed
on a FACSCalibur, and data were analyzed using CellQuest software (Becton Dickinson).
Results
Patient characteristics and clinical outcomes after allogeneic NM-HSCT.
The characteristics of twelve patients treated with NM-HSCT from HLA matched donors
are shown in Table 1. Eleven of the twelve patients had >50% bone marrow involvement with
CLL at the time of NM-HSCT, and one patient (UPN 18802) was transplanted in CR after
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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receiving chemotherapy for Richter’s transformation. Seven of the 12 patients achieved or
maintained a CR three to twelve months after transplant. One of these seven patients (UPN 9661)
only achieved a CR after receiving a donor leukocyte infusion (DLI) for the treatment of
Epstein-Barr virus associated lymphoproliferative disease. One additional patient (UPN 29449)
has an ongoing partial remission (PR) with a reduction in bone marrow tumor burden from 70%
pre transplant to 5% at six months after transplant. Six of these eight patients with a major
antitumor response developed acute GVHD and five developed extensive chronic GVHD. Four
patients had persistent or progressive CLL after NM-HSCT, despite the administration of DLI to
one of these patients (UPN 28196). All four non-responding patients developed acute GVHD,
and one of three that survived beyond day 100 developed chronic GVHD. One of the
non-responding patients remains alive with persistent CLL. There was no difference in the
immunosuppressive therapy regimens used for GVHD prophylaxis between responding and
non-responding patients.
Generation of CLL-reactive T cells from post-transplant PBMC
We hypothesized that donor cell engraftment might result in the development of a T cell
response specific for recipient minor H or tumor-associated antigens on CLL and contribute to
tumor regression. Aliquots of recipient CLL that were cryopreserved pre-transplant were used to
stimulate T cells obtained from the recipient post-transplant after donor engraftment was
established. CLL expresses class I and II MHC molecules and can be recognized by CD8+ and
CD4+ T cells, but has low levels of costimulatory and adhesion molecules (19, 20). Engagement
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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of CD40 on CLL with CD40 ligand (CD40L) upregulates the expression of co-stimulatory
(CD80 and CD86), adhesion (CD54 and CD58) and MHC molecules and makes the tumor cells
more efficient APC (19, 20, 26). Therefore, we co-cultured the pre-transplant CLL from the
eleven patients with a high tumor burden with tCD40L prior to their use in vitro for stimulating
post-transplant T cells. The frequency of CLL cells in the cultures after CD40L activation was
30.1-97.4% (median 80%) as assessed by co-expression of CD5 and CD19. In the patient in
morphologic CR prior to HSCT, we expanded CD5+ CD19+ CLL cells from bone marrow cells
after multiple stimulations with tCD40L and used these cells as APC. CLL activated through
CD40 expressed higher levels of co-stimulatory, adhesion, and MHC molecules as previously
reported (data not shown) (19, 20, 26).
PBMC were obtained from each recipient at intervals after HSCT, stimulated twice one
week apart with γ-irradiated recipient CD40-CLL, and then evaluated for lysis of recipient
CD40-CLL, and recipient and donor B-LCL. CD40-CLL rather than thawed primary CLL cells
were used as target cells in these experiments because CD40-CLL were of higher purity and
exhibited improved viability and uptake of 51Cr. Cytotoxic T lymphocytes (CTL) that recognized
both recipient CLL and B-LCL, but not donor B-LCL were detected after two stimulations at one
or more time points from all seven patients with a CR after HSCT or DLI, and from the one
patient with a major partial response at six months after HSCT (Figure 1A). Stimulation of
PBMC obtained directly from each of the donors with recipient CD40-CLL under the same
conditions did not elicit specific CTL activity against recipient CLL or B-LCL (data not shown),
demonstrating the reactivity detected in post-transplant PBMC resulted from in vivo activation of
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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T cells to recipient minor H and possibly tumor-associated antigens.
The antitumor activity of NM-HSCT is often delayed for several months after transplant
and we found the kinetics with which CLL-reactive T cell responses developed after HSCT
differed in individual patients. CLL-reactive T cells developed rapidly after transplant in some
patients including UPN 22388 (Figure 1A), who had a circulating lymphocyte count of 28,500
cells/μl consisting of >90% CD5+ CD19+ tumor cells at the time of transplant. CLL-reactive T
cells were detected in this patient in the PBMC sample obtained on day 35 post-transplant, and
coincided with a dramatic reduction in CD19+ CD5+ B cells. T cell responses specific for CLL
were not detected in other patients until several months after transplant, after improvement in
donor CD3 T cell chimerism to >90% (UPN 24487; UPN 25843) or reduction in
immunosuppressive drug therapy (UPN 21899) (Figure 1A). All four of the patients who did not
achieve CR after transplantation lacked specific cytotoxicity for recipient CLL at any of the time
points analyzed (Figure 1B), despite the occurrence of GVHD in all patients and >90% donor
CD3 T cell chimerism in three of the four patients.
The development of GVHD in the non-responding patients indicates that alloreactive T
cells must be present in these patients, but our assays suggest the alloreactivity was not directed
against antigens on malignant cells. An alternative explanation is that there was a defect in APC
function of the CLL cells from the nonresponding patients. To address this possibility, we
evaluated the ability of CD40-CLL from the four non-responding patients and one responding
patient to elicit alloreactive T cells in a mixed lymphocyte culture using T cells from an MHC
disparate unrelated donor. We found that the CLL from the non-responding patients were
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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equivalently effective APC (Figure 2A, B). Additionally, a non-responding patient (UPN 28196)
and a responding patient (UPN 22388) were both HLA-A2+ males, and the CLL from each of
these patients stimulated IFN-γ production from a T cell clone specific for the SMCY minor H
antigen (FIDSYICQV) (Figure 2C). These data demonstrate the failure to detect CLL-reactive T
cells in non-responders was not due to lack of APC function by the tumor cells, but rather
suggest that the specificity of donor alloreactive T cells that caused GVHD in the recipient was
not directed against minor H antigens expressed on recipient CLL.
CD4+ and CD8+ T cells that recognize CLL develop in responding patients
Analysis of the phenotype of the CLL-reactive T cell lines generated from each of the
eight responding patients showed the presence of both CD4+ and CD8+ T cells in cultures from
all of the 8 patients. To determine whether both T cell subsets recognized CLL, we stimulated an
aliquot of the polyclonal cultures derived from each patient with recipient CD40-CLL, and
evaluated IFN-γ production. As shown for two representative patients, a fraction of the CD4+ and
CD8+ T cells from each patient produced IFN-γ after stimulation with recipient CLL and B-LCL,
but not with donor B-LCL, demonstrating recipient minor H antigens on CLL were recognized
by both CD4+ and CD8+ T cells (Figure 3A, B). To determine whether both CD4+ and CD8+ T
cells also lysed CLL, we sorted T cells that produced IFN-γ from two patients into CD4+ and
CD8+ fractions and expanded each subset by stimulation with anti-CD3 monoclonal antibody.
After a single cycle of expansion, the CD4+ IFN-γ+ fraction contained 96.2 and 99.8% CD4+ T
cells respectively, and the CD8+ IFN-γ+ fraction contained 74.1% and 96.2% CD8+ T cells
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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respectively (data not shown). We used these enriched populations as effector cells against
recipient CD40-CLL, primary CLL, and recipient and donor B-LCL. The CD4+ T cells from
each patient exhibited absent or weak lytic activity against recipient CD40-CLL and B-LCL, and
failed to lyse primary CLL cells (Figure 3C). By contrast, the CD8+ T cells from each patient
efficiently lysed both recipient CD40-stimulated and primary CLL (Figure 3D), and lysis was
inhibited by anti class I monoclonal antibody (data not shown). Donor B-LCL and CD40
activated B cells served as controls and were not lysed by CD8+ T cells.
Clonal analysis of the CD8+ T cell response to CLL reveals recognition of both minor
histocompatibility and tumor-associated antigens.
All of the patients who responded to NM-HSCT also developed GVHD, which is
mediated by donor T cells specific for minor H antigens expressed on cells in the skin,
gastrointestinal tract, and/or liver (30-32). Minor H antigens have previously been identified that
are preferentially expressed on hematopoietic cells including B-lineage cells, and recognition of
these antigens may be associated with less GVHD (30, 33-35). Thus, the isolation of T cell
clones from CLL-reactive T cell lines derived from our patients would provide reagents to
identify additional minor H antigens that could potentially be targeted to induce a GVL response
without GVHD. We focused our efforts on analysis of the CD8+ T cell response because the
development of minor H antigen-specific CD8+ T cells after DLI has correlated with tumor
regression in other settings (33, 36), and CD8+ T cells that produced IFN-γ in response to
CD40-CLL lysed primary CLL cells more efficiently than CD4+ T cells (Figure 3C, D).
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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We derived CD8+ T cell clones from T cell lines generated at time points that coincided
with tumor regression from 7 responding patients. In three patients where adequate numbers of
cryopreserved CLL was available, γ-irradiated CLL rather than anti-CD3 monoclonal antibody
was used for stimulation in the cloning cultures. In the four patients where tumor cells were
limited, cloning wells positive for growth were screened for recognition of recipient and donor
B-LCL to identify clones specific for minor H antigens. These T cell clones were subsequently
analyzed for recognition of recipient CD40-CLL and fibroblasts to identify minor H antigens that
were expressed on tumor cells and preferentially on hematopoietic cells. In the three patients
where we had sufficient tumor cells for cloning, we tested each positive well for recognition of
recipient CLL and recipient and donor B-LCL to identify T cell clones specific for either minor
H or tumor-associated antigens.
Multiple CD8+ T cell clones that recognized a minor H antigen presented by both
recipient CD40 CLL and B-LCL, but not donor B-LCL were isolated from all 7 patients (11 for
UPN 22388; 10 for UPN 9661; 6 for UPN 21899; 2 for UPN 24487; 12 for UPN 18802; 6 for
UPN 28736; 13 for UPN 29449). We attempted to map the HLA-restricting allele for each minor
H antigen-specific T cell clone by testing recognition of a panel of B-LCL from unrelated donors
that shared only a single HLA allele with the recipient. The restricting HLA allele and an
estimate of the allele frequency was determined for 9 minor H antigen-specific T cell clones
(Table 2A). Three of these T cell clones also lysed IFN-γ pre-treated fibroblasts (>10% lysis)
demonstrating the expression of the minor H antigen recognized by these T cells was not
restricted to hematopoietic cells. Five clones did not lyse fibroblasts and were specific for minor
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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H antigens that are potentially selectively expressed on hematopoietic cells (Table 2A). We were
unable to map the restricting HLA-allele for many of the T cell clones because the panel of
partially matched unrelated B-LCL was not sufficient for conclusive assignment. However, based
on differential recognition of B-LCL from unrelated donors, we could conclude that the T cell
lines from 6 of the 7 patients targeted at least two distinct minor H antigens. Thus, at the time of
tumor regression after NM-HSCT, the T cell response was directed against multiple distinct
minor H antigens expressed on CLL.
UPN 22388 had a dramatic reduction in circulating tumor cells early after transplant and
did not develop GVHD in the first 80 days (Figure 4A). Eleven T cell clones were isolated from
blood obtained on day 63 and all were restricted by HLA-B40 as shown for clone 2A2 (Table
2A). These T cell clones did not recognize dermal fibroblasts consistent with the absence of
clinical GVHD in this patient at day 63 after HSCT. The patient subsequently developed
persistent chronic GVHD at day 120 suggesting that the T cell response might have broadened to
include recognition of additional minor H antigens. T cell clones were generated from blood
obtained at day 120 and 730 after transplant when the patient had active chronic GVHD, and
analysis of the HLA-restricting allele for 9 of these T cell clones revealed recognition of
additional distinct minor H antigens presented by HLA-A2, HLA-A31, HLA-B15, HLA-B40 and
HLA-Cw3 (Table 2B). In contrast to the HLA-B40-restricted T cell clone isolated at day 63 that
did not recognize fibroblasts, the minor H antigens targeted by 5 of the T cell clones isolated at
these later time points were expressed both by CD40 CLL and fibroblasts. One clone (13C6)
isolated at day 120 only lysed B-LCL from male HLA-A2+ donors suggesting it was specific for
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
18
a minor H antigen encoded on the Y-chromosome. Further studies revealed that 13C6 recognized
a previously described epitope encoded by the SMCY gene (Figure 4B, C), that has been
associated with GVHD in prior studies (30). Of interest, SMCY-specific T cells were not
detected in the T cell lines generated at day 63 by tetramer analysis when the patient did not have
GVHD, but tetramer binding cells were readily detected in the T cell lines generated from day
120 and 374 samples (Figure 4D). These data demonstrate that additional minor H
antigen-specific T cells are recruited with time after NM-HSCT, analogous to epitope spreading
that has been described in autoimmunity and potentially induced by cytotoxic tumor cell death
(37, 38). The T cell clones that were specific for broadly expressed minor H antigens and isolated
late after NM-HSCT, also recognized the recipient’s CLL, and may contribute to sustaining the
GVL effect (Table 2B).
The percentage of CD8+ T cells that produced INF-γ was higher after stimulation with
CD40-CLL compared to recipient B-LCL in many patients, suggesting that T cells specific for
minor H antigens or tumor-associated antigens that are selectively expressed on CLL may be
present. In 3 patients in which sufficient tumor cells were available to use both as stimulator cells
during cloning and as target cells in screening assays, multiple T cell clones that recognized
recipient CLL cells but not B-LCL were identified (Table 2C). The proportion of T cell clones
that lysed recipient CLL but not B-LCL varied between 30 and 79% of all T cell clones isolated
from these patients, suggesting that the tumor-specific response was a significant component of
the overall T cell response to CLL. We tested 16 clones obtained from the 3 patients against
panels of primary and activated CLL from other patients sharing at least one HLA-allele to
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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determine if these T cell clones might be directed against shared and/or non-shared
tumor-associated antigens. Twelve of the 16 clones only lysed primary and/or activated CLL
from the particular patient they were derived, but not CLL from other patients. Four clones
generated from UPN 22388 (14D1, 8A9, 10A10, 30B2) recognized CLL from one or more
unrelated patients, however we did not have large enough panels of CLL to conclusively identify
the HLA-restricting allele (data not shown). Nevertheless, our data demonstrate that T cells
specific for antigens exclusively expressed on CLL but not on B-LCL are elicited after
NM-HSCT. These antigens may include non-shared or shared tumor-specific antigens on CLL
cells and/or minor H antigens that are only expressed on CLL.
Discussion
NM-HSCT can result in a durable remission for 40-70% of CLL patients who progress
after fludarabine based regimens (11, 15-17). The efficacy of HSCT in CLL does not correlate
with factors that determine outcome after standard chemotherapy and is ascribed to a GVL effect
mediated by immune cells in the stem cell graft (9, 10, 13, 14). This study is the first to analyze
the presence and kinetics of development of CLL-reactive T cells after NM-HSCT and to
correlate T cell responses with antitumor efficacy. The results demonstrate that T cells that
specifically recognize recipient CLL and B-LCL, but not donor B-LCL develop in all patients
who achieved or maintained an antitumor response after NM-HSCT, but not in patients with
persistent or progressive disease.
We used recipient CD40-CLL rather than PBMC as APC since our focus was to uncover
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
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T cells specific for antigens on the tumor cells, and the number of T cells in post-transplant
samples was not sufficient to compare the alloreactivity that would be elicited using recipient
APC derived from different cell types. In all responding patients both CD8+ and CD4+ T cells
that recognized recipient CLL and B-LCL, but not donor B-LCL were detected by IFN-γ
production and/or cytotoxicity demonstrating that a component of the T cell response that
developed in responding patients is directed against minor H antigens on CLL. We used
CD40-CLL cells as target cells in the initial assays to assess tumor cell recognition by the
polyclonal T cell lines generated from each patient. In subsequent experiments, CD4+ and CD8+
T cells were selectively enriched from the T cell lines and assessed for recognition of primary
CLL cells. CD8+ T cells efficiently lysed primary CLL cells demonstrating that tumor cell
recognition by this subset did not depend on CD40 activation. Isolation of individual CD8+ T cell
clones from the polyclonal T cell lines confirmed that a significant component of the antitumor
response was specific for minor H antigens, some of which were not broadly expressed on
nonhematopoietic cells. Studies are in progress to clone the genes that encode minor H antigens
recognized by these CLL-reactive CD8+ T cells.
A greater frequency of T cells produced IFN-γ in response to recipient CLL than to
recipient B-LCL, suggesting that nonpolymorphic tumor-associated antigens expressed only by
CLL may be recognized. This was confirmed in a subset of three patients where sufficient tumor
cells were available to use as APC in the cloning cultures. CD8+ T cell clones that lysed recipient
CLL but not B-LCL were isolated from all three patients. The tumor-associated antigens
recognized by most of the T cell clones were not shared by CLL cells from unrelated individuals,
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
21
suggesting these antigens are derived from mutations or protein sequences that are unique to the
CLL of the individual patient. Autologous CLL-reactive T-cells have been described previously
(39), and immunoglobulin idiotype has been identified as a tumor antigen in B cell malignancies
(40, 41). The possibility that idiotype is being recognized by a component of the T cell response
elicited after NM-HSCT is being currently being investigated.
The results of our analysis of CLL-reactive T cells after NM-HSCT provide insight into
why the GVL effect may be so potent in eradication of this malignancy. Efforts to target
malignancies by T cell immunotherapy are commonly directed at a single tumor-associated
antigen, and can fail because of the outgrowth of antigen loss tumor variants (42, 43). Our
studies show that the CLL-reactive T cell response after allogeneic NM-HSCT is comprised of
both CD8+ and CD4+ T cells, and commonly directed against multiple antigens. Moreover,
analysis of the CD8+ T cell response over time in a single patient illustrated the emergence of
new T cell clones specific for minor H antigens expressed by CLL. This diversification of the
alloreactive T cell response capable of recognizing CLL may diminish the potential for
outgrowth of tumor cell variants, and may explain why complete tumor regression is often
delayed for several months.
Stimulation of post-transplant PBMC with recipient CD40-CLL did not elicit
CLL-reactive T cells from any patient with persistent or progressive disease after transplant,
despite GVHD in these patients. The failure of the GVL effect in these patients may be due to the
absence of disparity between donors and recipients in the subset of minor H antigens that are
expressed by CLL, or to a failure of T cells specific for disparate minor H antigens expressed on
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
22
CLL to be activated and expand in vivo. CLL cells have been demonstrated to induce defects in
T cells in tumor bearing patients and to be defective in immune synapse formation (44, 45), and
it is possible that a large tumor burden at the time of transplant reduces the activation of donor T
cells specific for antigens present on CLL.
Acute or chronic GVHD developed in all of the patients who achieved a CR,
emphasizing the difficulty separating the potent GVL effect from GVHD with current
approaches to NM-HSCT. Clonal analysis of CLL-reactive CD8+ T cells that developed in
patients with GVHD demonstrated recognition of minor H antigens that are broadly expressed on
nonhematopoietic tissues (30, 32). Prior studies have identified minor H antigens such as
PANE-1 and HB-1 that are selectively expressed on B-lineage cells (34, 35), but the allele
frequency of these antigens is such that very few patients could benefit from targeted therapy to
induce a GVL effect. Discovery of additional determinants with selective expression on recipient
hematopoietic cells may allow targeting of leukemic cells by adoptive T cell transfer or
vaccination. Ideally, nonpolymorphic antigens expressed on CLL might be used as targets for
immunotherapy. There is evidence that immunoglobulin framework, survivin, and fibromodulin
peptides can be presented by CLL (46-48). Further studies to identify tumor-specific and minor
H antigens that are recognized by T cells that develop after NM-HSCT might lead to more
effective and selective targeting of malignant cells after NM-HSCT.
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Titles and legends to figures
Figure 1. Cytotoxicity of T cell lines generated from NM-HSCT recipients by stimulation
with recipient CD40-CLL. PBMC were obtained from recipients at the designated days after
NM-HSCT, stimulated twice one week apart with γ-irradiated recipient CD40-CLL, and tested in
a cytotoxicity assay against Cr51-labeled recipient CD40-CLL (■), recipient B-LCL (■) and
donor B-LCL (□). Data is shown for an effector to target ratio of 30:1 or 40:1. A. Reactivity of
T cell lines generated from patients who had a major antitumor response after NM-HSCT. B.
Reactivity of T cell lines generated from patients who had persistent or progressive disease after
NM-HSCT.
Figure 2. CD40-CLL from patients who fail to respond to nonmyeloablative HSCT are
effective antigen presenting cells. A-B. PBMC from an unrelated HLA disparate individual
(JKW) were stimulated in a mixed lymphocyte culture twice one week apart with γ-irradiated
CD40-CLL derived from a responding patient (UPN 22388) as a control (A), and with
γ-irradiated CD40-CLL from each of the 4 transplant recipients who failed to achieve a CR after
transplant (B). The T cell lines were then assayed for cytotoxic activity against autologous
(JKW) B-LCL, recipient CD40-CLL and recipient B-LCL if available at effector to target ratios
of 1:1 – 100:1. C. CLL cells and B-LCL from an HLA-A2+ male patients who had progressive
CLL after NM-HSCT (28196) or who responded to NM-HSCT (22388) were tested for the
ability to stimulate an SCMY-specific T cell clone (CTL) to produce IFN-γ. T cells and
stimulator cells were co-cultured at a ratio of 3:1 for 24 hours, supernatants were collected and
IFN-γ was measured by ELISA.
CLL-REACTIVE T CELL RESPONSES AFTER STEM CELL TRANSPLANT
28
Figure 3. CLL-reactive CD4+ and CD8+ T cells that produce IFN-γ and cytolytic CD8+ T
cells are elicited after NM-HSCT in responding patients. A, B. T cell lines from two
representative patients (UPN 22388 and UPN 9661) were stimulated with recipient CD40-CLL
(top panels) and B-LCL (middle panels), and with donor B-LCL (lower panels) and stained with
PE-conjugated IFN-γ detection reagent and either FITC-conjugated anti-CD4 or anti-CD8
monoclonal antibodies. C, D. CD4+ and CD8+ T cells were isolated by IFN-γ+ capture after
stimulation with recipient CD40-CLL, expanded with anti-CD3 monoclonal antibody and tested
for lysis of recipient B-LCL, CD40-CLL and primary CLL cells, and against donor B-LCL and
CD40 activated B cells. Data is shown at an effector to target ratio of 30:1.
Figure 4. CD8+ T cells reactive with the SMCY peptide FIDSYICQV develop late after
NM-HSCT in UPN 22388. A. Decline in total lymphocyte count in UPN 22388 in the first 28
days post-transplant. B. Recognition of HLA-A*0201 LCL from unrelated female and male
donors by CD8+ T cell clone 13C6 isolated from PBMC obtained at day 120 after allogeneic
NM-HSCT. The data is shown for an E/T of 10:1. C. Recognition by SMCY-specific clone 13C6:
recipient B-LCL ( ♦ ); donor B-LCL either unpulsed (■) or pulsed ( ) with 10 μM
FIDSYICQV and 3 μg/ml human β-2 microglobulin. D. SMCY-specific T cells developed late
after transplant in UPN 22388. The T cell lines generated from UPN 22388 at day +63, +120,
and +374 after transplant and the T cell clone 13C6 were stained with PE-conjugated anti CD3
and FITC conjugated anti CD8 monoclonal antibodies and an APC conjugated HLA-A*0201
tetramer folded with FIDSYICQV.